APR January/February 2022 - 75

« INSTRUMENTATION
release batch material, and to perform other analytical assays, such as
potency assays. Both qPCR and ddPCR platforms have been applied
to determining AAV genome titer.3,5,6
qPCR has several disadvantages
for this application including the requirement of a well calibrated
standard curve and the influence of PCR bias on quantitative results.
Standard curves for qPCR-based methods could be linearized plasmid
containing the target sequence or a reference standard AAV with a
defined genome titer.6
One of the additional challenges is that qPCR
assays can have a wide lab-to-lab variability.7 The dependence of
a qPCR-based method on a standard can also be challenging if that
standard is changed throughout the lifecycle of the method or a
program; particularly if the method has been used for dosing in clinical
trials and the change of standard results in different quantitation such
as shown in the example in Table 1.
Table 1. Change in plasmid standard may result in different
genome titer quantitation. Two different preparations or lots of the
same plasmid resulted in different genome titer results.
Sample
1
2
3
Plasmid
Standard
Preparation A
Result (VG/mL)
8.40E+12
8.94E+12
3.60E+12
Plasmid
Standard
Preparation B
Result (VG/mL)
2.86E+12
3.04E+12
1.24E+12
Fold Difference
2.94
2.94
2.91
expresses AAV2 rep and cap genes. These cells are co-infected with
AAV and adenovirus type 5 (Ad5), resulting in the replication of
infectious AAV particles within the cells. Replicated AAV can then be
detected by PCR and infectious titer is calculated using the SpearmanKärber
method. Utilizing endpoint dilutions, single infection events
can be detected using the TCID50 method.8
The generated result is an
absolute value represented as TCD50/mL or infectious units per mL
(IU/mL). While the assay can show biological activity of AAV particles, it
has several drawbacks. Different AAV capsids will have varying tropism
to the HeLaRC32 cells used in the assay system and the absolute value
result may not truly be representative of how infectious an AAV vector
is. Additionally, the absolute value result can be quite variable. This
leads to reproducibility challenges within the same lab and between
labs, making assay transfer difficult.7
The replicative ability of the
AAV vector in the assay system is also not reflective of what occurs
when the vector is administered in vivo, as recombinant AAV vectors
for gene therapy are replication incompetent. Therefore, assays that
measure cellular uptake or internalization via PCR may be more
relevant for AAV therapies. Additionally, relative internalization assays
which measure the AAV transgene relative to a housekeeping gene
would have a higher level of precision and robustness as opposed to
an absolute measurement assay. Furthermore, the TCID50 assay does
not demonstrate vector-specific potency either at the expression or
functional level. Program-specific assays are required for AAV products
as they advance into clinical trials and this necessitates development
of additional functional assays to support AAV gene therapy programs.
How the standard is quantified for its value assignment can also impact
the quantitative results it generates. For example, Table 2 shows a
linearized plasmid standard quantified in ng/µL by two common
dsDNA detection methods, PicoGreen assay and UV absorbance by
NanoDrop. Each method generates different values, which when
converted to a copies per volume value, have a significant difference.
Because of the challenges associated with qPCR methods, ddPCRbased
assays are being more widely adopted. They have proved to be
highly precise and provide absolute quantitation, rather than relying
on a standard curve.3,5
Table 2. Comparison of plasmid standard curve
copy number determination.
Measured by Concentration (ng/µL) Copies/µL % Difference
PicoGreen Assay
UV Absorbance
12.40
15.85
1.83E+09
2.34E+09
24.4
PCR for Expression and Potency
(RT-PCR, RT-ddPCR)
Potency of a gene therapy product is dependent, in part, on how
well the transgene is expressed. Therefore, measuring transgene
expression levels in transduced cells is an important analytical method
for all gene therapy products. PCR-based methods can be used to
detect
transgene RNA expression levels for many types of gene
Infectivity
A platform approach to determining one attribute of AAV vector activity
is to measure its infectious titer or infectivity. This type of method can
be applied to virtually any AAV vector. The most common method for
determining AAV infectivity is a 50% tissue culture infectious dose
(TCID50) assay. TCID50 utilizes a stable cell line, HeLaRC32, which
therapies such as gene-augmentation and gene-silencing therapies.
For gene-augmentation, the goal is to replace a missing or aberrant
protein within the cell to modulate the disease state. The transgene
typically contains a coding DNA sequence that gets transcribed into
mRNA and translated into functional protein. In vitro expression of a
gene-augmentation transgene is often quantified using RT-qPCR and/
or RT-ddPCR for mRNA, or an immunoassay for protein.
In gene-silencing gene therapies, the transgene cassette expresses
a small RNA, such as a miRNA or siRNA, to reduce the expression of
an endogenous gene in the target cells to modulate the disease
state. Analytical PCR methods may be used to quantitate expression
levels of the small RNA expressed from the AAV genome, as well as
measure the reduction of target mRNA levels. The most common PCRbased
method for small RNA detection uses stem-loop primer reverse
transcription followed by PCR quantitation.9
For gene-silencing strategies that involve knockdown of an
endogenous gene target, quantitating the downregulation of target
www.americanpharmaceuticalreview.com |
| 75
»
http://www.americanpharmaceuticalreview.com

APR January/February 2022

Table of Contents for the Digital Edition of APR January/February 2022

APR January/February 2022 - Cover1
APR January/February 2022 - Cover2
APR January/February 2022 - 1
APR January/February 2022 - 2
APR January/February 2022 - 3
APR January/February 2022 - 4
APR January/February 2022 - 5
APR January/February 2022 - 6
APR January/February 2022 - 7
APR January/February 2022 - 8
APR January/February 2022 - 9
APR January/February 2022 - 10
APR January/February 2022 - 11
APR January/February 2022 - 12
APR January/February 2022 - 13
APR January/February 2022 - 14
APR January/February 2022 - 15
APR January/February 2022 - 16
APR January/February 2022 - 17
APR January/February 2022 - 18
APR January/February 2022 - 19
APR January/February 2022 - 20
APR January/February 2022 - 21
APR January/February 2022 - 22
APR January/February 2022 - 23
APR January/February 2022 - 24
APR January/February 2022 - 25
APR January/February 2022 - 26
APR January/February 2022 - 27
APR January/February 2022 - 28
APR January/February 2022 - 29
APR January/February 2022 - 30
APR January/February 2022 - 31
APR January/February 2022 - 32
APR January/February 2022 - 33
APR January/February 2022 - 34
APR January/February 2022 - 35
APR January/February 2022 - 36
APR January/February 2022 - 37
APR January/February 2022 - 38
APR January/February 2022 - 39
APR January/February 2022 - 40
APR January/February 2022 - 41
APR January/February 2022 - 42
APR January/February 2022 - 43
APR January/February 2022 - 44
APR January/February 2022 - 45
APR January/February 2022 - 46
APR January/February 2022 - 47
APR January/February 2022 - 48
APR January/February 2022 - 49
APR January/February 2022 - 50
APR January/February 2022 - 51
APR January/February 2022 - 52
APR January/February 2022 - 53
APR January/February 2022 - 54
APR January/February 2022 - 55
APR January/February 2022 - 56
APR January/February 2022 - 57
APR January/February 2022 - 58
APR January/February 2022 - 59
APR January/February 2022 - 60
APR January/February 2022 - 61
APR January/February 2022 - 62
APR January/February 2022 - 63
APR January/February 2022 - 64
APR January/February 2022 - 65
APR January/February 2022 - 66
APR January/February 2022 - 67
APR January/February 2022 - 68
APR January/February 2022 - 69
APR January/February 2022 - 70
APR January/February 2022 - 71
APR January/February 2022 - 72
APR January/February 2022 - 73
APR January/February 2022 - 74
APR January/February 2022 - 75
APR January/February 2022 - 76
APR January/February 2022 - 77
APR January/February 2022 - 78
APR January/February 2022 - 79
APR January/February 2022 - 80
APR January/February 2022 - 81
APR January/February 2022 - 82
APR January/February 2022 - 83
APR January/February 2022 - 84
APR January/February 2022 - Cover3
APR January/February 2022 - Cover4
https://www.nxtbookmedia.com